CONFORMATION OF MANGANESE(II)-NUCLEOTIDE COMPLEXES BOUND TO RABBIT MUSCLE CREATINE-KINASE - C-13 NMR MEASUREMENTS USING [2-C-13]ATP AND [2-C-13]ADP

Conformations of cation-nucleotide complexes bound to rabbit muscle cr eatine kinase were investigated by measuring paramagnetic effects on C -13 spin relaxation in E . Mn[2-C-13]ATP and E . Mn[2-C-13]ADP at thre e different frequencies, viz., 50, 75, and 125 MHz, and as a function of temperature in the range of 7-35 degrees C (at 75 MHz). Arrhenius p lots of the temperature dependencies of relaxation rates show a positi ve slope with low activation energies of 1.3 +/- 0.2 kcal/mol and 2.0 +/- 0.2 kcal/mol for E . Mn ATP acid E . MnADP, respectively. The rela xation rates of both complexes show strong frequency dependence, indic ating that these rates are not exchange limited, Analysis of the data yields Mn(II)-2C distances of 10.0 +/- 0.5 Angstrom for E . MnADP and 8.6 +/- 0.5 Angstrom for E . MnADP. These data were interpreted, along with previously published information, on the location of the cation with respect to the phosphate chain [Jarori, G. K., Ray, B. D., & Nage swara Rao, B. D. (1985) Biochemistry 24, 3487-3494], and on the adenos ine conformation [Murali, N., Jarori, G., K., & Nageswara Rao, B. D., (1993) Biochemistry 32, 12941-12948] in these complexes. The Mn(II)-2C distances depend on the orientation of the phosphate chain relative t o the adenosine moiety. Conformational searches were performed by vary ing the two torsion angles, phi(1)(C-4'-C-5'-O-5'-P-alpha), and phi(2) (C-5'-O-5'-P-alpha-O-alpha beta), along with CHARMm energy computation s, in order to determine acceptable conformations compatible with the distances determined. The significant difference in the Mn(II)-2C dist ances in E . MnATP and E . MnADP is indicative of the structural alter ations occurring at the active site as the enzyme turns over.